The Nef protein of HIV-1 is essential for high-titer HIV replication and for AIDS progression. Nef binds and activates members of the Src protein-tyrosine kinase family, namely Hck, Lyn, and c-Src. Hck is expressed primarily in macrophages, a major site of HIV-1 replication, while Lyn and c-Src are expressed in all HIV-1 susceptible cell types. Accumlating evidence shows that Nef-induced Src family kinase (SFK) activation is important for HIV replication and disease progression. Recently, we identified a novel compound (DFP-4-aminopropanol) that inhibits Nef-mediated Hck activity in vitro as well as Nef-dependent HIV-1 replication in cell culture. In Aim 1, I propose to examine the efficacy of this compound and its structural analogs for anti-retroviral activity in other HIV-1 target cells (T-cells, PBMCs and macrophages) and against HIV-1 variants carrying different alleleic variants of Nef. I will also test the compounds against strains of HIV-1 resistant to viral reverse transcriptase and protease inhibitors. In Aim 2, I will test the hypothesis that SFKs are the relevant targets for DFP-4-aminopropanol and related compounds. I will first explore the importance of Src kinases in HIV replication through RNAi-mediated knockdown of kinase expression. I will then engineer SFK mutants that are resistant to the inhibitory action of DFP-4- aminopropanol. If SFKs are direct targets for the compound, expression of these SFK mutants in U87MG cells should restore HIV-1 replication in the presence of the inhibitor. These experiments will implicate SFKs in HIV replication and demonstrate that DFP-4-aminopropanol works at least in part by blocking Nef- dependent SFK activation. In Aim 3, I propose to examine whether SFKs are direct targets for DFP-4- aminopropanol by biotinylating the compound, immobilizing it on strepavidin-agarose beads, and incubating it with either recombinant Nef:SFK complexes or cell lysates from the U87MG HIV replication system and other HIV target cells where the compound is active. Bound proteins will be identified via tryptic fingerprinting/mass spectrometry to determine whether SFKs interact directly with DFP-4-aminopropanol. Additionally, other proteins that bind to the immobilized compound will be identified via these proteomic techniques, which may provide novel insight into mechanisms by which Nef supports HIV replication and identify additional targets for drug discovery. PUBLIC HEALTH RELEVANCE: There is a critical need for discovery of new anti-HIV drug targets due to the rising incidence of drug resistance of clinically-relevant HIV strains. This compound represents the first to target a viral protein:host cell protein kinase complex, providing a new lead for anti-HIV drug development.